Method and apparatus for preventing cross-examination of biochemical test wells in a microtiter test plate

ABSTRACT

This invention relates to an improved method and apparatus for preventing cross-contamination of biochemical test wells included in a microtiter test plate which comprises the covering of the biochemical test wells with a porous barrier that has been impregnated with an approximately 10% (W/V) solution of a solid, water soluble compound and then dried before use, the impregnating material being one selected form a group of chemical compounds consisting of inorganic acid phosphates, the acid salts of amino acids and the polycarboxylic organic acids which are relatively non-volatile, non-hygroscopic and non-toxioc to humans and bacteria while remaining effective to neutralize alkaline gases without attacking the barrier material.

This is a continuation-in-part of my copending application Ser. No. 723,360 filed Apr. 15, 1985 in which I disclosed a method for neutralizing volatile alkaline products evolved through bacterial growth taking place in the wells of a combined biochemical identification-antiobiotic microtiter test plate, thus preventing false biochemical test results due to cross-contamination with the volatile alkaline products which consisted of impregnating a porous barrier with an acidic compound capable of neutralizing the alkaline products and then covering the test wells with such a barrier. At the time, I discovered that potassium dihydrogen phosphate was an ideal acidic compoun for impregnation of the test well barrier and it continues to be one of the best, if not, in fact, the best one to use. It possesses all the properties I have found desirable in the acidic compound to be used for this purpose such as, for example, those which follow.

Ideally, the acidic compound which will be effective as an impregnate for the test well barrier should be readily soluble in water (at least 10% W/V) at or near ambient temperatures. Obviously, it should possess the ability to quickly and easily neutralize alkaline gases like ammonia while at the same time having a low toxicity for both humans and bacteria. Low volatility and being relatively non-hygroscopic are other desirable properties as well as that of not attacking the barrier material.

Bearing in mind the aforementioned properties, certain acidic compounds can be eliminated at the outset. For instance, strong acids like hydrochloric, nitric and sulfuric, in addition to being liquids under normal conditions of pressure and temperature, would have a damaging effect on the filter paper or other porous barrier. Hydrochloric acid is also too volatile to be of any use since it would evaporate while the barrier impregnated therewith was being dried. Other acids like hydrocyanic are too toxic for both humans and many types of bacteria to be practical. Other acids, such as boric, oxalic, and 5-sulfosalicylic, for example, are too irritating to the skin and mucous membrances to receive serious consideration. Acids such as adipic, salicylic, phthalic, and others of this type have a rather low solubility in water. Consequently, it would not be possible to impregnate the barrier with a sufficient concentration thereof to effectively neutralize the evolved alkaline products even though they would meet the other standards. Those acidic compounds that are highly hygroscopic in the dehydrated form like, for instance, glycine hydrochloride, should not be used since they pick up water from the test wells and form droplets that can fall back into the well giving false positive acid reactions and false negative alkaline reactions. Last, but by no means least, are the considerable number of acidic compounds such as acetic acid, propionic acid, butyric acid and the like which, while otherwise suitable, are either in the gaseous or liquid form at normal temperatures and pressures and, therefore, unsuitable because they cannot be used in the preparation of dry barriers that can be handled conveniently.

I have now discovered that, despite the several varied and distinctive properties that a suitable acidic impregnate should possess, there are several other acidic compounds in addition to sodium or potassium acid phosphate which are capable of neutralizing the alkaline products evolved by reasons of the bacterial growth taking place in the test wells of a microtiter test plate. For example, I have been able to show that, in addition to the inorganic acid phosphates, the acid salts of amino acids perform quite well as do the polycarboxylic and sulfonated organic acids.

It is, therefore, the principal object of the present invention to provide a broadened class of acidic compounds which can be satisfactorily impregnated into a porous medium and used to neutralize alkaline products evolved by bacterial growth taking place in the wells of a microtiter test plate, the presence of which would otherwise contaminate the biochemical test wells and possibly result in both false-positive and false-negative test results.

A second objective is the provision of acidic compounds of the class aforementioned which are essentially non-toxic to both humans and the bacteria growing in the wells.

Another object of the invention herein disclosed and claimed is one of providing a select group of compounds for the aforementioned purpose which are sufficiently soluble such that a quantity thereof in excess of that required to neutralize the volatile alkaline products evolved can be impregnated into the porous medium and remain there after it is dried.

Still another objective is to provide an acidic compound which, in dehydrated form, is non-hygroscopic to the extent that it is incapable of absorbing enough water from the suspension in the test well to produce a contaminated droplet on the underside of the acid-impregnated barrier that can drop off and reenter the well.

An additional object is to provide a group of chemical compounds for neutralizing volatile alkaline products that are water-soluble dry solids at ordinary temperatures and pressures and, therefore, can be dissolved in water and impregnated into a porous paper matte or similar material and dried without losing activity.

Further objects of the present invention are to provide a number of acidic compounds useful in the neutralization of volatile alkaline products that are readily obtainable on the open market, safe to handle, have a good shelf life and are not so strong as to destroy or even damage the porous material into which they are introduced.

Other objects will be in part apparent and in part pointed out specifically hereinafter in connection with the detailed description which follows.

I found that, in my attempt to select acidic compounds which satisfied the several criteria mentioned above that would be required of the ideal impregnate to neutralize volatile alkaline products evolved by bacterial growth in microtiter and biochemical test wells, there were, as a matter of fact, very few possibilities. Those I selected for testing were as follows:

1. Boric acid

2. Oxalic acid

3. 5-Sulfosalicylic acid

4. Potassium acid phthalate

5. Glycine hydrochloride

6. Cysteine hydrochloride

7. Bis-tris buffer (effective pH range 5.8 to 7.2)

8. Mes buffer (effective pH range 5.5 to 6.7)

9. DL Malic acid

10. Malonic acid

11. DL Tartaric acid

12. Citric acid

Even some of the above acidic compounds possess one or more undesirable properties such as, for example, low solubility; yet, I tested them anyway.

The initial tests were run using 10% (W/V) solutions except for potassium acid phthalate (8%), boric acid (5%) and oxalic acid (9%) due to the limited solubility of the latter compounds. The porous member chosen for impregnation was Whatman #1 filter paper cut into rectangles sized to cover the so-called "gram-negative" biochemical test wells in a minimum inhibitory concentration (MIC)identification microplate. Inocula were prepared according to the generally accepted procedure for MIC plates to be used for antibiotic susceptibility testing. The following test organisms were used as the inocula in the tests although not all were necessarily tested in each experiment:

1. Pseudomonas aeruginosa

2. Citrobacter freundii

3. Aeromonas caviae

4. Pasteurella aerogenes

5. Acinetobacter anitratus

6. Providencia alcalifaciens

7. Klebsiella pneumoniae

8. Proteus mirabilis

All of the tests performed utilized Pseudomonas aeruginosa as the inoculum in all of the MIC wells because of its capacity to produce ammonia along with its resistance to most of the antibiotics. All of the biochemical test wells were then inoculated with one of the test organisms listed above. A strip impregnated with a 15% potassium dihydrogen phosphate solution was placed over the biochemical test wells of a second plate and the experimental test strips were used on the additional plates. Since it is well known that certain tests must be conducted under anaerobic conditions, the medium contained in these wells was covered with mineral oil in accordance with accepted practice. Specifically, the oxidation-fermentation of glucose test well; the wells for testing the decarboxylation of arginine, lysine, and ornithine; and the well for detecting urease activity are usually covered. In these experiments, the oxidation-fermentation of glucose well was always covered with oil while the other wells mentioned above were sometimes covered with oil and sometimes not. Since ammonia will cause false positive reactions in the unprotected decarboxylase and urease test wells, the mineral oil was omitted with some of the test organisms in order to put the barrier strips to a more critical test.

All of the plates were incubated at 36° C. for 18 to 22 hours before the first evaluation was made. The same impregnated barriers were then replaced on the plates and they were again observed after a total incubation time of approximately 40 to 42 hours. Prolonged incubation is sometimes necessary in order to properly identify the test organism. In my experiments, however, I subjected the plates to prolonged incubation for the specific purpose of determining which of the protective strips would remain effective for the longest time.

Results of the tests were evaluated on the basis of comparisons of the reactions that the organisms tested were known to produce with conventional tests, i.e. using other than microtiter test plates; the reactions observed using microplates with unprotected biochemical test wells; and those in which the biochemical test wells were covered with the neutralizing barriers impregnated with the acidic compounds being evaluated. Barrier strips impregnated with potassium dihydrogen phosphate were included because I had already thoroughly tested this neutralizing agent and knew it to be quite effective; therefore, it provided me with a standard against which to evaluate the effectiveness of the other compounds being tested.

In the tables which follow, abbreviations are used to identify the various biochemical substrates and they are:

    ______________________________________                                         Abbr.  Substrate        Abbr.     Substrate                                    ______________________________________                                         OF/G   Fermentation of Glucose                                                                         GLU       Glucose                                      MAN    Mannitol         ARA       Arabinose                                    TRE    Trehalose        CEL       Cellobiose                                   SOR    Sorbitol         MEL       Melibiose                                    SUC    Sucrose          RAF       Raffinose                                    RHA    Rhamnose         ADO       Adonitol                                     ARG    Arginine         LYS       Lysine                                       ORN    Ornithine        URE       Urea                                         CIT    Citrate          MAL       Malonate                                     ______________________________________                                    

Also, as previously noted, two of the acidic compounds tested were MES buffer and BIS-TRIS buffer. The MES buffer is (2 [N-Morpholine] ethanesulfonic acid, whereas, the BIS-TRIS buffer is [Bis (2-hydroxyethyl) imino tris (hydroxymethyl) methane.

                  TABLE I                                                          ______________________________________                                         Test Organism - Acinetobacter anitratus                                        Incubation                        Tartaric Acid                                Time    No Barrier    KH.sub.2 PO.sub.4 (15%)                                                                    (10%)                                        ______________________________________                                         *23 hours                                                                              False neg. on Okay        Okay                                                 ARA, CEL, MEL                                                                  and RHA                                                                *42 hours                                                                              False neg. on +/- on CEL  False neg.                                           GLU, ARA, CEL,            on CEL                                               MEL and RHA                                                            ______________________________________                                    

                  TABLE II                                                         ______________________________________                                         Test organism - Acinetobacter anitratus                                                                          5-                                           Incubation                        Sulfosalicylic                               Time    No Barrier    KH.sub.2 PO.sub.4 (15%)                                                                    Acid (10%)                                   ______________________________________                                         *23 hours                                                                              False neg. on Okay        Okay                                                 ARA, CEL, MEL                                                                  and RHA                                                                *42 hours                                                                              False neg. on +/- on CEL  False neg.                                           GLU, ARA, CEL,            on CEL                                               MEL AND RHA                                                            **191/2 False neg. on False pos. on                                                                              False pos.                                   hours   ARA and CEL   ARG, LYS and                                                                               on ARG,                                              False pos. on ORN         LYS and                                              ARG, LYS, ORN             ORN                                                  and URE                                                                **42 hours                                                                             False neg. on +/- on CEL  False pos.                                           GLU, ARA, CEL,                                                                               False pos. on                                                                              on LYS                                               MEL and RHA   LYS                                                              False pos. on                                                                  ARG, LYS, ORN                                                                  and URE                                                                ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                        **These tests were conducted with only the OF/G wells oiled with mineral       oil.                                                                     

                  TABLE III                                                        ______________________________________                                         Test organism - acinetobacter anitratus                                        Incubation                         Malic Acid                                  Time    No Barrier     KH.sub.2 PO.sub.4 (15%)                                                                    (10%)                                       ______________________________________                                         *21 hours                                                                              False neg. on  Okay        Okay                                                CEL +/- on MEL                                                         *41 hours                                                                              False neg. on  +/- on CEL  +/- on                                              GLU, ARA, CEL, and GLU     CEL                                                 MEL and RHA                                                            ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE IV                                                         ______________________________________                                         Test organism - acinetobacter anitratus                                                                           Malonic                                     Incubation                         Acid                                        Time    No Barrier     KH.sub.2 PO.sub.4 (15%)                                                                    (10%)                                       ______________________________________                                         *21 hours                                                                              False neg. on  Okay        Okay                                                CEL +/- on MEL                                                         *41 hours                                                                              False neg. on  +/- on CEL  Okay                                                GLU, ARA, CEL,                                                                 MEL and RHA                                                            **191/2 False neg. on  False pos. on                                                                              False pos.                                  hours   ARA and CEL    ARG, LYS and                                                                               on ARG,                                             False pos. on  ORN         LYS and                                             ARG, LYS, ORN              ORN                                                 and URE                                                                **42 hours                                                                             False neg. on  +/- on CEL  False pos.                                          GLU, ARA, CEL, False pos. on                                                                              on LYS                                              MEL and RHA    LYS                                                             False pos. on                                                                  ARG, LYS, ORN                                                                  and URE                                                                **19 hours                                                                             False neg. on  False pos. on                                                                              False pos.                                          CEL +/- on ARA ARG and ORN on ARG                                              False pos. on              and ORN                                             ARG, ORN and URE                                                       **42 hours                                                                             False neg. on  Okay        Okay                                                GLU, ARA and MEL                                                               False pos. on                                                                  ARG, ORN and URE                                                       **191/2 False neg. on CEL                                                                             False pos. on                                                                              False pos.                                  hours   +/- on ARA     ARG and ORN on ARG                                              and MEL                    and ORN                                             False pos. on                                                                  ARG, ORN and URE                                                       **41 hours                                                                             False neg. on  False neg. on                                                                              False neg.                                          GLU, ARA, MEL  CEL         on CEL                                              CEL and RHA                                                                    False pos. on                                                                  ARG, ORN and URE                                                       ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                        **These tests were conducted with only the OF/G wells oiled with mineral       oil.                                                                     

                                      TABLE V                                      __________________________________________________________________________     Test Organism - acinetobacter anitratus                                        Incubation Time                                                                         No Barrier                                                                               KH.sub.2 PO.sub.4 (15%)                                                                  Oxalic Acid (9%)                                  __________________________________________________________________________     *21 hours                                                                               False neg. on                                                                            Okay      Okay                                                       CEL +/- on MEL                                                        *41 hours                                                                               False neg. on                                                                            +/- on GLU and                                                                           False neg. on                                              GLU, ARA, CEL                                                                            CEL       CEL +/- on GLU                                             MEL and RHA                                                           __________________________________________________________________________

                                      TABLE VI                                     __________________________________________________________________________     Test Organism - acinetobacter anitratus                                        Incubation Time                                                                         No Barrier KH.sub.2 PO.sub.4 (15%)                                                                Cysteine.HCl 10%                                   __________________________________________________________________________     **191/2 hours                                                                           False neg. on                                                                             False pos. on                                                                          False pos. on                                               ARA and CEL                                                                               ARG, LYS and                                                                           ARG, LYS and                                                False pos. on                                                                             ORN     ORN                                                         ARG, LYS, ORN                                                                  and URE                                                               **42 hours                                                                              False neg. on                                                                             +/- on CEL                                                                             False neg. on CEL                                           GLU, ARA, CEL                                                                             False pos. on                                                                          +/- on GLU, RHA                                             MEL and RHA                                                                               LYS     and ORN                                                     False pos. on      False pos. on                                               ARG, LYS, ORN      LYS                                                         and URE                                                               __________________________________________________________________________      *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                        **These tests were conducted with only the OF/G wells oiled with mineral       oil.                                                                     

                                      TABLE VII                                    __________________________________________________________________________     Test Organism - acinetobacter anitratus                                        Incubation Time                                                                         No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                Glycine.HCl 10%                                  __________________________________________________________________________     **191/2 hours                                                                           False neg. on CEL                                                                           False pos. on                                                                          False pos. on                                             +/- on ARA and MEL                                                                          ARG and ORN                                                                            ARG and ORN                                               False pos. on                                                                  ARG, ORN and URE                                                      **41 hours                                                                              False neg. on                                                                               False neg. on                                                                          False neg. on                                             GLU, ARA, MEL                                                                               CEL     GLU and CEL                                               CEL and RHA                                                                    False pos. on                                                                  ARG, ORN and URE                                                      __________________________________________________________________________

                                      TABLE VIII                                   __________________________________________________________________________     Test organism - pasteurella aerogenes                                                                         5-Sulfosalicylic                                Incubation Time                                                                         No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                  Acid 10%                                        __________________________________________________________________________     **191/2 hours                                                                           False neg. on                                                                              Okay      Okay                                                     GLU and ARA                                                                    +/- on SUC                                                                     False pos. on                                                                  ARG, LYS, ORN,                                                                 CIT and MAL                                                           **42 hours                                                                              False neg. on                                                                              False neg on SUC                                                                         False neg. on                                            GLU, ARA and SUC                                                                           +/- on GLU                                                                               GLU and SUC                                              False pos. on                                                                              False pos. on MAL                                                  ARG, LYS, ORN,                                                                             +/- on ORN                                                         CIT and MAL                                                           __________________________________________________________________________      **These tests were conducted with only the OF/G wells oiled with mineral       oil.                                                                     

                                      TABLE IX                                     __________________________________________________________________________     Test organism - pasteurella aerogenes                                          Incubation Time                                                                         No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                Malic Acid 10%                                    __________________________________________________________________________     *21 hours                                                                               False neg. on                                                                              Okay    Okay                                                       GLU, ARA and SUC                                                               False pos. on                                                                  CIT and MAL                                                           *41 hours                                                                               False neg. on                                                                              False neg. on                                                                          +/- on GLU and                                             GLU, ARA and SUC                                                                           GLU and ARA                                                                            SUC                                                        False pos. on                                                                              False pos. on                                                      CIT and MAL MAL                                                       __________________________________________________________________________      *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE X                                                          ______________________________________                                         Test organism - pasteurella aerogenes                                                                               Malon-                                    Incubation                           Acid                                      Time    No Barrier     KH.sub.2 PO.sub.4 (15%)                                                                      (10%)                                     ______________________________________                                         *21 hours                                                                              False neg. on  Okay          Okay                                              GLU, ARA and SUC                                                               False pos. on                                                                  CIT and MAL                                                            *41 hours                                                                              False neg. on  False neg. on Okay                                              GLU, ARA and SUC                                                                              GLU and SUC                                                     False pos. on  False pos. on                                                   CIT and MAL    MAL                                                     **191/2 False neg. on  Okay          Okay                                      hours   GLU and ARA                                                                    +/- on SUC                                                                     False pos. on                                                                  ARG, LYS, ORN,                                                                 CIT and MAL                                                            **42 hours                                                                             False neg. on  False neg. on SUC                                                                            Okay                                              GLU, ARA and SUC                                                                              +/- on GLU                                                      False pos. on  False pos. on MAL                                               ARG, LYS, ORN  +/- on ORN                                                      CIT and MAL                                                            **191/2 False neg. on  Okay          Okay                                      hours   ARA and SUC                                                                    +/- on GLU False                                                               pos. on ARG, LYS,                                                              ORN, CIT and MAL                                                       **41 hours                                                                             False neg. on  +/- on MAL    Okay                                              GLU, ARA and SUC                                                                              False pos. on                                                   False pos. on  ORN                                                             ARG, LYS, ORN,                                                                 CIT and MAL                                                            ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                        **These tests were conducted with only the OF/G wells oiled with mineral       oil.                                                                     

                  TABLE XI                                                         ______________________________________                                         Test organism - pasteurella aerogenes                                                                             Oxalic                                      Incubation                         Acid                                        Time    No Barrier     KH.sub.2 PO.sub.4 (15%)                                                                    (9%)                                        ______________________________________                                         *21 hours                                                                              False neg. on  Okay        Okay                                                GLU, ARA and SUC                                                               False pos. on                                                                  CIT and MAL                                                            *41 hours                                                                              False neg. on  False neg. on                                                                              +/- on                                              GLU, ARA and SUC                                                                              GLU and SUC GLU                                                 False pos. on  False pos. on                                                   CIT and MAL    MAL                                                     ______________________________________                                    

                                      TABLE XII                                    __________________________________________________________________________     Test organism - pasteurella aerogenes                                          Incubation Time                                                                         No Barrier KH.sub.2 PO.sub.4 (15%)                                                                  Cysteine.HCl (10%)                               __________________________________________________________________________     **191/2 hours                                                                           False neg. on                                                                             Okay      Okay                                                      GLU and ARA                                                                    +/- on SUC                                                                     False pos. on                                                                  ARG, LYS, ORN,                                                                 CIT and MAL                                                           **42 hours                                                                              False neg. on                                                                             False neg on SUC                                                                         False neg. on                                             GLU, ARA and SUC                                                                          +/- on GLU                                                                               GLU and SUC                                               False pos. on                                                                             False pos. on MAL                                                                        False pos. on                                             ARG, LYS, ORN,                                                                            +/- on ORN                                                                               MAL and ORN                                               CIT and MAL                                                           __________________________________________________________________________      *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                        **These tests were conducted with only the OF/G wells oiled with mineral       oil.                                                                     

                  TABLE XIII                                                       ______________________________________                                         Test organism - pasteurella aerogenes                                          Incubation             KH.sub.2 PO.sub.4                                                                        Glycine.HCl                                   Time    No Barrier     (15%)     (10%)                                         ______________________________________                                         **191/2 False neg. on  Okay      Okay                                          hours   ARA and SUC                                                                    +/- on GLU                                                                     False pos. on                                                                  ARG, LYS, ORN,                                                                 CIT and MAL                                                            **41 hours                                                                             False neg. on  +/- on    False neg.                                            GLU, ARA and SUC                                                                              MAL False on GLU                                                False pos. on  pos. on   +/- on SUC                                            ARG, LYS, ORN, ORN                                                             CIT and MAL                                                            ______________________________________                                    

                  TABLE XIV                                                        ______________________________________                                         Test organism - providencia alcalifaciens                                                                         Malic Acid                                  Incubation Time                                                                           No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                    (10%)                                       ______________________________________                                         *21 hours  False neg.  Okay        Okay                                                   on ADO                                                                         +/- on GLU                                                                     False pos. on                                                                  CIT and MAL                                                         *41 hours  False neg.  False pos.  Okay                                                   on ADO      on MAL                                                             False pos.                                                                     on MAL                                                              ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                        **These tests were conducted with only the OF/G wells oiled with mineral       oil.                                                                     

                  TABLE XV                                                         ______________________________________                                         Test organism - providencia alcalifaciens                                                                           Malon-                                    Incubation                           ic Acid                                   Time    No Barrier       KH.sub.2 PO.sub.4 (15%)                                                                    (10%)                                     ______________________________________                                         *21 hours                                                                              False neg.       Okay        Okay                                              on ADO                                                                         +/- on GLU                                                                     False pos. on                                                                  CIT and MAL                                                            *41 hours                                                                              False neg.       False pos.  Okay                                              on ADO           on MAL                                                        False pos.                                                                     on MAL                                                                 *191/2  +/- on GLU       Okay        Okay                                      hours   and ADO                                                                        False pos. on                                                                  MAL, ARG, LYS,                                                                 ORN and URE                                                            *42 hours                                                                              +/- on ADO       +/- on MAL  Okay                                              False pos. on                                                                  MAL, ARG, LYS,                                                                 ORN and URE                                                            *19 hours                                                                              False neg. on    Okay        Okay                                              ADO +/- on GLU                                                                 False pos. on                                                                  MAL, ARG, LYS,                                                                 ORN and URE                                                            *42 hours                                                                              +/- on ADO       +/- on MAL  Okay                                              False pos. on                                                                  MAL, ARG, LYS,                                                                 ORN and URE                                                            *191/2  +/- on GLU and ADO                                                                              Okay        Okay                                      hours   False pos. on                                                                  MAL, ARG, LYS,                                                                 ORN and URE                                                            *41 hours                                                                              False neg. on    +/- on MAL  Okay                                              GLU and ADO                                                                    False pos. on                                                                  MAL, ARG, LYS,                                                                 ORN and URE                                                            ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XVI                                                        ______________________________________                                         Test organism - providencia alcalifaciens                                      Incubation                       Oxalic Acid                                   Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    (9%)                                          ______________________________________                                         *21 hours                                                                              False neg.   Okay        Okay                                                  on ADO                                                                         +/- on GLU                                                                     False pos. on                                                                  CIT and MAL                                                            *41 hours                                                                              False neg.   False pos.  Okay                                                  on ADO       on MAL                                                            False pos.                                                                     on MAL                                                                 ______________________________________                                    

                  TABLE XVII                                                       ______________________________________                                         Test organism - providencia alcalifaciens                                      Incubation             KH.sub.2 PO.sub.4                                                                        5-Sulfosalicylic                              Time    No Barrier     (15%)     Acid 10%                                      ______________________________________                                         *191/2  +/- on GLU     Okay      False pos. on                                 hours   and ADO                  ARG, LYS and                                          False pos. on            ORN                                                   MAL, ARG, LYS,                                                                 ORN and URE                                                            *42 hours                                                                              +/- on ADO     +/- on    Okay                                                  False pos. on  MAL                                                             MAL, ARG, LYS,                                                                 ORN and URE                                                            ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XVIII                                                      ______________________________________                                         Test organism - providencia alcalifaciens                                      Incubation                        Cysteine.HCl                                 Time    No Barrier    KH.sub.2 PO.sub.4 (15%)                                                                    10%                                          ______________________________________                                         *191/2  +/- on GLU    Okay        False pos. on                                hours   and ADO                   ARG, LYS                                             False pos. on             and ORN                                              MAL, ARG, LYS,                                                                 ORN and URE                                                            *42 hours                                                                              +/- on ADO    +/- on MAL  Okay                                                 False pos. on                                                                  MAL, ARG, LYS,                                                                 ORN and URE                                                            ______________________________________                                    

                                      TABLE XIX                                    __________________________________________________________________________     Test organism - providencia alcalifaciens                                      Incubation Time                                                                         No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                Glycine.HCl 10%                                  __________________________________________________________________________     *191/2 hours                                                                            +/- on GLU and ADO                                                                          Okay    Okay                                                      False pos. on                                                                  MAL, ARG, LYS,                                                                 ORN and URE                                                           *41 hours                                                                               False neg. on                                                                               +/- on MAL                                                                             Excess moisture                                           GLU and ADO          on strip                                                  False pos. on        Acid drip into                                            MAL, ARG, LYS,       wells                                                     ORN and URE                                                           __________________________________________________________________________      *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XX                                                         ______________________________________                                         Test organism - escherichia coli                                               Incubation                       Tartaric Acid                                 Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    10%                                           ______________________________________                                         *24 hours                                                                              False pos.   Okay        Okay                                                  on CIT                                                                         and MAL                                                                *42 hours                                                                              False neg.   Okay        Okay                                                  on RHA                                                                         +/- on MAN                                                                     False pos. on                                                                  CIT and MAL                                                            ______________________________________                                    

                  TABLE XXI                                                        ______________________________________                                         Test organism - escherichia coli                                               Incubation                       5-Sulfosalicylic                              Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    Acid 10%                                      ______________________________________                                         *24 hours                                                                              False pos.   Okay        Okay                                                  on CIT                                                                         and MAL                                                                *42 hours                                                                              False neg.   Okay        Okay                                                  on RHA                                                                         +/- on MAN                                                                     False pos. on                                                                  CIT and MAL                                                            ______________________________________                                    

                  TABLE XXII                                                       ______________________________________                                         Test organism - escherichia coli                                               Incubation                       Oxalic Acid                                   Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    9%                                            ______________________________________                                         *24 hours                                                                              False pos.   Okay        Okay                                                  on CIT                                                                         and MAL                                                                *42 hours                                                                              False neg.   Okay        Okay                                                  on RHA                                                                         +/- on MAN                                                                     False pos. on                                                                  CIT and MAL                                                            ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXIII                                                      ______________________________________                                         Test organism - escherichia coli                                                                                  Citric Acid                                 Incubation Time                                                                           No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                    10%                                         ______________________________________                                         *24 hours  False pos.  Okay        Okay                                                   on CIT                                                                         and MAL                                                             *42 hours  False neg.  Okay        Okay                                                   on RHA                                                                         +/- on MAN                                                                     False pos. on                                                                  CIT and MAL                                                         ______________________________________                                    

                  TABLE XXIV                                                       ______________________________________                                         Test organism - escherichia coli                                               Incubation                      Potassium Acid                                 Time    No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                    Phthalate 8%                                   ______________________________________                                         *24 hours                                                                              False pos.  Okay        False pos.                                             on CIT                  on MAL                                                 and MAL                                                                *42 hours                                                                              False neg.  Okay        False pos.                                             on RHA                  on CIT and MAL                                         +/- on MAN                                                                     False pos. on                                                                  CIT and MAL                                                            ______________________________________                                    

                  TABLE XXV                                                        ______________________________________                                         Test organism - escherichia coli                                               Incubation                       MES Buffer                                    Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    10%                                           ______________________________________                                         *24 hours                                                                              False pos.   Okay        False pos.                                            on CIT                   on CIT                                                and MAL                  and MAL                                       *42 hours                                                                              False neg.   Okay        False pos.                                            on RHA                   on CIT                                                +/- on MAN               and MAL                                               False pos. on                                                                  CIT and MAL                                                            ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXVI                                                       ______________________________________                                         Test organism - escherichia coli                                                                                  Boric Acid                                  Incubation Time                                                                           No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                    5%                                          ______________________________________                                         *24 hours  False pos.  Okay        False pos.                                             on CIT                  on CIT                                                 and MAL                 and MAL                                     *42 hours  False neg.  Okay        False pos.                                             on RHA                  on CIT                                                 +/- on MAN              and MAL                                                False pos. on                                                                  CIT and MAL                                                         ______________________________________                                    

                  TABLE XXVII                                                      ______________________________________                                         Test organism - citrobacter freundii                                           Incubation                        Tartaric Acid                                Time    No Barrier    KH.sub.2 PO.sub.4 (15%)                                                                    10%                                          ______________________________________                                         *20 hours                                                                              False pos.    Okay        Okay                                                 on ARG, LYS,                                                                   ORN and MAL                                                            *41 hours                                                                              False neg.    Okay        Okay                                                 on OF/G                                                                        False pos. on                                                                  ARG, LYS, ORN,                                                                 URE and MAL                                                            ______________________________________                                    

                  TABLE XXVIII                                                     ______________________________________                                         Test organism - citrobacter freundii                                                                             5-                                           Incubation                        Sulfosalicylic                               Time    No Barrier    KH.sub.2 PO.sub.4 (15%)                                                                    Acid 10%                                     ______________________________________                                         *20 hours                                                                              False pos.    Okay        Okay                                                 on ARG, LYS,                                                                   ORN and MAL                                                            *41 hours                                                                              False neg.    Okay        +/- on                                               on OF/G                   GLU                                                  False pos. on                                                                  ARG, LYS, ORN,                                                                 URE and MAL                                                            ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXIX                                                       ______________________________________                                         Test organism - citrobacter freundii                                           Incubation             KH.sub.2 PO.sub.4                                                                       Oxalic Acid                                    Time    No Barrier     (15%)    9%                                             ______________________________________                                         *20 hours                                                                              False pos.     Okay     Okay                                                   on ARG, LYS,                                                                   ORN and MAL                                                            *41 hours                                                                              False neg.     Okay     False pos. on                                          on OF/G                 ARG, LYS and                                           False pos. on           ORN                                                    ARG, LYS, ORN,                                                                 URE and MAL                                                            ______________________________________                                    

                  TABLE XXX                                                        ______________________________________                                         Test organism - citrobacter freundii                                           Incubation                         Citric Acid                                 Time    No Barrier     KH.sub.2 PO.sub.4 (15%)                                                                    10%                                         ______________________________________                                         *20 hours                                                                              False pos.     Okay        Okay                                                on ARG, LYS,                                                                   ORN and MAL                                                            *41 hours                                                                              False neg.     Okay        Okay                                                on OF/G                                                                        False pos. on                                                                  ARG, LYS, ORN,                                                                 URE and MAL                                                            ______________________________________                                    

                  TABLE XXXI                                                       ______________________________________                                         Test organism - citrobacter freundii                                           Incubation             KH.sub.2 PO.sub.4                                                                       Potassium Acid                                 Time    No Barrier     (15%)    Phthalate 8%                                   ______________________________________                                         *20 hours                                                                              False pos.     Okay     Okay                                                   on ARG, LYS,                                                                   ORN and MAL                                                            *41 hours                                                                              False neg.     Okay     +/- on ARA                                             on OF/G                 and RHA                                                False pos. on           False pos.                                             ARG, LYS, ORN,          on MAL                                                 URE and MAL                                                            ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXXII                                                      ______________________________________                                         Test organism - citrobacter freuhdii                                           Incubation                        MES Buffer                                   Time    No Barrier    KH.sub.2 PO.sub.4 (15%)                                                                    10%                                          ______________________________________                                         *20 hours                                                                              False pos.    Okay        Okay                                                 on ARG, LYS,                                                                   ORN and MAL                                                            *41 hours                                                                              False neg.    Okay        False pos.                                           on OF/G                   on MAL                                               False pos. on                                                                  ARG, LYS, ORN,                                                                 URE and MAL                                                            ______________________________________                                    

                  TABLE XXXIII                                                     ______________________________________                                         Test organism - citrobacter freundii                                           Incubation                         Boric Acid                                  Time    No Barrier     KH.sub.2 PO.sub.4 (15%)                                                                    5%                                          ______________________________________                                         *20 hours                                                                              False pos.     Okay        False pos.                                          on ARG, LYS,               on MAL                                              ORN and MAL                                                            *41 hours                                                                              False neg.     Okay        False pos.                                          on OF/G        on MAL                                                          False pos. on                                                                  ARG, LYS, ORN,                                                                 URE and MAL                                                            ______________________________________                                    

                  TABLE XXXIV                                                      ______________________________________                                         Test organism - aeromonas caviae                                               Incubation                       Tartaric Acid                                 Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    10%                                           ______________________________________                                         *20 hours                                                                              False pos.   Okay        Okay                                                  on LYS, ORN,                                                                   URE, CIT                                                                       and MAL                                                                *41 hours                                                                              False pos.   False pos.  False pos.                                            on LYS, ORN, on CIT      on CIT                                                URE, CIT                                                                       and MAL                                                                ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXXV                                                       ______________________________________                                         Test organism - aeromonas caviae                                               Incubation                       5-Sulfosalicylic                              Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    Acid 10%                                      ______________________________________                                         *20 hours                                                                              False pos.   Okay        Okay                                                  on LYS, ORN,                                                                   URE, CIT                                                                       and MAL                                                                *41 hours                                                                              False pos.   False pos.  Okay                                                  on LYS, ORN, on CIT                                                            URE, CIT                                                                       and MAL                                                                ______________________________________                                    

                  TABLE XXXVI                                                      ______________________________________                                         Test organism - aeromonas caviae                                               Incubation                       Oxalic Acid                                   Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    9%                                            ______________________________________                                         *20 hours                                                                              False pos.   Okay        Okay                                                  on LYS, ORN,                                                                   URE, CIT                                                                       and MAL                                                                *41 hours                                                                              False pos.   False pos.  False pos.                                            on LYS, ORN, on CIT      on CIT                                                URE, CIT                 False neg.                                            and MAL                  on ARG                                        ______________________________________                                    

                  TABLE XXXVII                                                     ______________________________________                                         Test organism - aeromonas caviae                                                                                  Citric Acid                                 Incubation Time                                                                           No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                    10%                                         ______________________________________                                         *20 hours  False pos.  Okay        Okay                                                   on LYS, ORN,                                                                   URE, CIT                                                                       and MAL                                                             *41 hours  False pos.  False pos.  False pos.                                             on LYS, ORN,                                                                               on CIT      on CIT                                                 URE, CIT                                                                       and MAL                                                             ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXXVIII                                                    ______________________________________                                         Test organism - aeromonas caviae                                               Incubation                       Potassium Acid                                Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    Phthalate 8%                                  ______________________________________                                         *20 hours                                                                              False pos.   Okay        Okay                                                  on LYS, ORN,                                                                   URE, CIT                                                                       and MAL                                                                *41 hours                                                                              False pos.   False pos.  False pos.                                            on LYS, ORN, on CIT      on CIT                                                URE, CIT                 and MAL                                               and MAL                                                                ______________________________________                                    

                  TABLE XXXIX                                                      ______________________________________                                         Test organism - aeromonas caviae                                               Incubation                       MES Buffer                                    Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    10%                                           ______________________________________                                         *20 hours                                                                              False pos.   Okay        Okay                                                  on LYS, ORN,                                                                   URE, CIT                                                                       and MAL                                                                *41 hours                                                                              False pos.   False pos.  False pos.                                            on LYS, ORN, on CIT      on CIT                                                URE, CIT                 and MAL                                               and MAL                                                                ______________________________________                                    

                  TABLE XXXX                                                       ______________________________________                                         Test organism - aeromonas caviae                                                                                  Boric Acid                                  Incubation Time                                                                           No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                    5%                                          ______________________________________                                         *20 hours  False pos.  Okay        False pos.                                             on LYS, ORN,            on MAL                                                 URE, CIT                                                                       and MAL                                                             *41 hours  False pos.  False pos.  False pos.                                             on LYS, ORN,                                                                               on CIT      on CIT                                                 URE, CIT                and MAL                                                and MAL                                                             ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXXXI                                                      ______________________________________                                         Test organism - pseudomonas aeruginosa                                         Incubation                       KH.sub.2 PO.sub.4 Plus                        Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    1% Gelatin                                    ______________________________________                                         *19 hours                                                                              False neg.   False pos. on                                                                              False pos. on                                         on GLU, ARA  LYS and ORN LYS and ORN                                           and MEL                                                                        False pos.                                                                     on LYS, ORN                                                                    and URE                                                                *42 hours                                                                              False neg.   False neg.  False neg.                                            on GLU, ARA, on ARG      on GLU                                                and MEL                  +/- on MEL                                            False pos.               False neg.                                            on LYS, ORN              on ARG                                                and URE                  False pos. on                                                                  LYS and ORN                                   ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXXXII                                                     ______________________________________                                         Test organism - pseudomonas aeruginosa                                         Incubation                       Malonic Acid                                  Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    10%                                           ______________________________________                                         *19 hours                                                                              False neg.   False pos. on                                                                              False pos. on                                         on GLU, ARA  LYS and ORN LYS and ORN                                           and MEL                                                                        False pos.                                                                     on LYS, ORN                                                                    and URE                                                                *42 hours                                                                              False neg.   False neg.  False neg.                                            on GLU, ARA, on ARG      on ARG                                                and MEL                  False pos.                                            False pos.               on LYS                                                on LYS, ORN                                                                    and URE                                                                *191/2  False neg.   False pos. on                                                                              False pos. on                                 hours   on GLU, ARA  LYS and ORN LYS and ORN                                           and MEL                                                                        False pos.                                                                     on LYS, ORN                                                                    and URE                                                                *41 hours                                                                              False neg.   False pos.  False pos.                                            on GLU, ARA, on LYS      on LYS                                                and MEL      and ORN     and ORN                                               False pos.                                                                     on LYS, ORN                                                                    and URE                                                                ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXXXIII                                                    ______________________________________                                         Test organism - pseudomonas aeruginosa                                                                         10%                                            Incubation                      Malonic Acid                                   Time    No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                    Plus 1% Gelatin                                ______________________________________                                         *19 hours                                                                              False neg.  False pos. on                                                                              False pos. on                                          on GLU, ARA LYS and ORN LYS and ORN                                            and MEL                                                                        False pos.                                                                     on LYS, ORN                                                                    and URE                                                                *42 hours                                                                              False neg.  False neg.  False neg. on                                          on GLU, ARA,                                                                               on ARG      GLU and ARG                                            and MEL                 False pos.                                             False pos.              on LYS                                                 on LYS, ORN                                                                    and URE                                                                ______________________________________                                    

                  TABLE XXXXIV                                                     ______________________________________                                         Test organism - pseudomonas aeruginosa                                         Incubation                       Glycine.HCl                                   Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    10%                                           ______________________________________                                         *191/2  False neg.   False pos. on                                                                              False pos. on                                 hours   on GLU, ARA  LYS and ORN LYS and ORN                                           and MEL                                                                        False pos.                                                                     on LYS, ORN                                                                    and URE                                                                *41 hours                                                                              False neg.   False pos.  False neg.                                            on GLU, ARA, on LYS      on GLU                                                and MEL      and ORN     +/- on RHA                                            False pos.               False pos. on                                         on LYS, ORN              LYS and ORN                                           and URE                                                                ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

                  TABLE XXXXV                                                      ______________________________________                                         Test organism - proteus mirabilis                                                                               15%                                           Incubation                       KH.sub.2 PO.sub.4 Plus                        Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    1% Gelatin                                    ______________________________________                                         *19 hours                                                                              False pos.   Okay        Okay                                                  on ARG, LYS                                                                    and MAL                                                                *42 hours                                                                              +/- on GLU   +/- on MAL  +/- on MAL                                            and TRE                                                                        False pos.                                                                     on ARG, LYS                                                                    and MAL                                                                ______________________________________                                    

                  TABLE XXXXVI                                                     ______________________________________                                         Test organism - proteus mirabilis                                              Incubation                       Malonic Acid                                  Time    No Barrier   KH.sub.2 PO.sub.4 (15%)                                                                    10%                                           ______________________________________                                         *19 hours                                                                              False pos.   Okay        Okay                                                  on ARG, LYS                                                                    and MAL                                                                *42 hours                                                                              +/- on GLU   +/- on MAL  Okay                                                  and TRE                                                                        False pos.                                                                     on ARG, LYS                                                                    and MAL                                                                ______________________________________                                    

                  TABLE XXXXVII                                                    ______________________________________                                         Test organism - proteus mirabilis                                                                              10%                                            Incubation                      Malonic Acid                                   Time    No Barrier  KH.sub.2 PO.sub.4 (15%)                                                                    Plus 1% Gelatin                                ______________________________________                                         *19 hours                                                                              False pos.  Okay        Okay                                                   on ARG, LYS                                                                    and MAL                                                                *42 hours                                                                              +/- on GLU  +/- on MAL  Okay                                                   and TRE                                                                        False pos.                                                                     on ARG, LYS                                                                    and MAL                                                                ______________________________________                                          *These tests were conducted with the OF/G, ARG, ORN, LYS and URE wells         oiled with mineral oil.                                                  

The preceding tests reveal the fact that boric acid, potassium acid phthalate, Bis-Tris buffer and Mes buffer are all unsuitable for use in treatment of the protective strips since each of them, while protecting some of the critical reactions, failed to protect others. I suspect that the two buffers, Bis-Tris buffer and Mes buffer both have pH ranges too high to provide adequate protection against the evolved alkaline gases under the conditions of these experiments. Boric acid, because of its low solubility in water, was used at the 15% (W/V) level for treating the paper strips and this could, perhaps, explain the low level of protection observed with some of the reactions.

The same sort of a solubility problem encountered with the boric acid also exists with potassium acid phthalate limiting its use to an 8% (W/V) concentration in water. Additionally, however, the relatively high pH (4.01) of the phthalic acid salt used to treat the strips when compared to potassium acid phosphate probably accounts for the significantly reduced protective action of the phthalate. Yet another factor is, of course, the significant difference in the molecular weights of these two acid salts (204 vs. 136) when comparing solutions of the same percentage concentration.

The paper strips impregnated with 5-sulfosalicylic acid provided adequate protection against the evolved alkaline products; however, as a practical matter, this acid proved to be too corrosive in that the barrier papers impregnated with it became brittle and essentially unusable after being stored for only a few months.

Glycine hydrochloride when used at a concentration of 10% (W/V) proved to be too hygroscopic. A high concentration of malonic acid (15%) also proved to be unsatisfactory for the same reason, i.e. excessive moisture take-up, when used as the impregnant and incubated for prolonged periods of time (40 to 42 hours). On the other hand, a 10% solution of malonic acid outperformed the potassium acid phosphate imprefnate even at the higher concentration of 15% (W/V) over extended incubation times of 40 to 42 hours in some of the reactions with some of the test organisms; whereas, during the shorter 18 to 22 hour incubation periods, the malonic acid and potassium acid phosphate impregnates showed little, if any significant differences.

All of the other compounds tested proved to be effective when used at concentrations of approximately 10% (W/V) including the oxalic acid at only 9%. Accordingly, oxalic acid, 5-sulfosalicylic acid, DL malic acid, DL tartaric acid and citric acid all proved effective at concentrations of around 10% (W/V). In addition to the forgoing list of polycarboxylic and sulfonated organic acids along with the previously tested inorganic acid phosphates, add cysteine hydrochloride which, of course, is an acid salt of an amino acid. All of the above have proven to be satisfactory impregnates to protect the most significant of the critical reactions in the biochemical test wells of a microtiter test plate against cross contamination resulting from evolved alkaline gases. 

What is claimed is:
 1. The improved method for preventing cross-contamination of bacterial test wells in a multiple-well antimicrobial test plate wherein certain of the wells contain growing cultures of bacteria of the type that evolve alkaline gases, which comprises: covering the wells with a porous membrane dried after having been impregnated with an approximately 10% (W/V) solution of a solid water-soluble compound selected from the group consisting of inorganic acid phosphates, the acid salts of amino acids and the polycarboxylic organic acids which are relatively non-volatile, non-hygroscopic and non-toxic to humans and bacteria.
 2. The improved method as set forth in claim 1 wherein the membrane is impregnated with an acid salt of an amino acid.
 3. The improved method as set forth in claim 1 wherein the membrane is impregnated with a polycarboxylic organic acid.
 4. The improved method as set forth in claim 1 in which the membrane is impregnated with cysteine hydrochloride.
 5. The improved method as set forth in claim 1 in which the membrane is impregnated with a polycarboxylic acid selected from the group consisting of citric acid, oxalic acid, malonic acid, DL malic acid and DL tartaric acid. 